June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Keratin 6a dampens Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by inhibiting ELKS/IKKε-dependent activation of NF-κB
Author Affiliations & Notes
  • Jonathan Chan
    Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
    Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Yan Sun
    Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Anand Bhushan
    Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Andy Lui
    Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Connie Tam
    Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
    Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Jonathan Chan None; Yan Sun None; Anand Bhushan None; Andy Lui None; Connie Tam None
  • Footnotes
    Support  NIH/NEI R01 EY030577 (CT), NIH/NEI P30EY025585(BA-A), Research to Prevent Blindness (RPB) Challenge Grant and Cleveland Eye Bank Foundation Grant.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 163. doi:
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    • Get Citation

      Jonathan Chan, Yan Sun, Anand Bhushan, Andy Lui, Connie Tam; Keratin 6a dampens Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by inhibiting ELKS/IKKε-dependent activation of NF-κB. Invest. Ophthalmol. Vis. Sci. 2023;64(8):163.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Corneal epithelial cells are important sentinels against ocular invading pathogens. Through TLR-mediated recognition of pathogen associated molecular patterns, these cells engage in NF-κB-dependent gene expression of antimicrobial peptides to directly ward off pathogens, as well as proinflammatory and chemotactic cytokines to promote immune cell infiltration. TLR-induced canonical ELKS-IKKα/β kinase complex phosphorylates the cytoplasmic inhibitor IκBα leading to its degradation and activation of NF-κB/RelA, whereas the non-canonical IKKε directly phosphorylates RelA at S468 to maximize its activity. We recently discovered that K6a, an abundant cytoskeletal factor in epithelial cells, downregulates TLR signaling. Here we investigate whether K6a represses both classes of IKKs.

Methods : Non-targeting or K6a-specific siRNA was transfected into human telomerase-immortalized cornea epithelial (hTCEpi) cells alone, or together with ELKS-specific or IKKε-specific siRNAs. Following transduction with NF-κB-luciferase adenovirus, cells were stimulated with P. aeruginosa culture supernatant for 0-24 h. Cell lysates were used for luciferase assay of NF-κB activation, immunoblotted for phosphorylated IKKα/β, IκBα, RelA and IKKε, or immunoprecipitated using anti-ELKS antibody followed by immunoblotting for K6a or IKKε. Culture media were collected for cytokine ELISA.

Results : K6a-KD hTCEpi cells had significantly increased NF-κb-luciferase activity and proinflammatory cytokine secretions (IL-1α, IL-8, CXCL1 and CCL20); IKKα/β phosphorylation and kinetics of IκBα degradation in these cells remained unchanged, whereas phosphorylation of IKKε (S172) and RelA (S468) increased. In contrast to K6a-KD cells, cells with double knockdown of K6a with ELKS or IKKε had reduced proinflammatory cytokine secretions. Remarkably, knocking down ELKS suppressed the stimulatory phosphorylation of IKKε and RelA. ELKS interacted with both K6a and IKKε.

Conclusions : Our studies demonstrated a specific role of cytoskeletal K6a in suppressing TLR/NF-κB pathway by inhibiting ELKS-dependent phosphorylation of IKKε and thereby RelA activation. The data thus revealed a previously unknown function of ELKS as an activator of non-canonical IKKε and its potential as a therapeutic target for treating bacterial keratitis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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